Q Beta replicase, an RNA-dependent RNA polymerase, is responsible for the replication of the genome of the RNA phage Q Beta. Two of the host-specified components of this complex enzyme are the Escherichia coli protein biosynthesis elongation factor EF-Tu and EF-Ts. These polypeptides act as the EF-Tu.Ts complex and do not perform their known protein synthetic functions in the RNA synthetic reaction. The experiments described here are designed to determine the relationship between structure and function in Q Beta replicase. In particular we are asking what functions the elongation factors are performing in Q Beta replicase and how they interact with the other enzyme subunits during the reaction. Using our denaturation/reconstitution system we will make and study Q Beta replicase with chemically altered, mutant, or heterologous EF-Tu.Ts. We will also utilize bifunctional protein and RNA cross-linking reagents to study changing subunit relationships during RNA synthesis. Because it seems reasonably likely that the EF-Tu.Ts in Q Beta replicase could be mimicking an unknown role played by this protein complex in uninfected cells, we will undertake a search for such an additional function for the EF-Tu.Ts complex. In so doing we will determine what percent of the elongation factors are in the complexed form and with what other cellular components they associate. Another host-coded polypeptide, host factor, required for in vitro Q Beta RNA replication is associated with the 30S ribosomal subunit in uninfected cells. We will perform experiments to determine its function in both protein synthesis and Q Beta RNA replication. Other projects include development of techniques to expand the number of templates which Q Beta replicase can transcribe; and purification and determination of the molecular mechanism of action of T-factor from yeast mitochondria.